Upon translocation of the peptidyl-tmRNA, the ribosome is reprogrammed to bind aminoacyl-tRNAs as specified by tmRNA. This process results in the covalent attachment of the tag peptide. Finally, when the "stop" codon of tmRNA is reached, the tagged protein and the tmRNA are released from the ribosome. Given that many of the methods developed for investigating tRNA, mRNA, and ribosomes are applicable to the study of tmRNA, answers to the most pressing questions about the functional role of tmRNA in bacterial metabolism are likely to be forthcoming. Recent studies identified three more proteases that degrade proteins tagged by tmRNA. One is the ATP-dependent, membrane-anchored zinc protease HflB, the only essential protease in Escherichia coli. Two others are the ATP-dependent cytosolic protease complexes ClpXP and ClpAP. The binding of tmRNA to ribosomes is much weaker than the analogous interaction with tRNA. To study ribosome tmRNA interactions, the authors applied cross-linking methods similar to those used successfully for investigating interactions between the ribosome, tRNA, and mRNA. In addition, the authors have demonstrated that ribosomes which lack protein S1 do not bind tmRNA. The recent progress in understanding the three-dimensional structure of the ribosome, when combined with the identification of cross-linked sites, will help us to analyze tmRNA interactions with the ribosome at the level of single nucleotide and amino acid residues. Furthermore, genetic and in vivo studies are expected to contribute significantly to our understanding of the complex interactions that might occur among tmRNA, the ribosome, and its various ligands.

E. coli tmRNA secondary structure with base pairs supported by comparative sequence analysis (Fox and Woese, 1975; Larsen and Zwieb, 1991). Perfect base pairs are connected with lines; G•U pairs are connected with open circles. Helices are highlighted in gray and numbered from 1 to 12 from the 5' end. Helical sections are given extensions with lowercase letters. The nucleosides are labeled with dots in increments of 10 and are numbered, if allowed by the available space. The 5'-to-3' direction of the single RNA chain is indicated by lines with open arrowheads. The four pseudoknots are marked pk1 to pk4. The solid star marks the beginning of the tmRNA coding region. In-frame stop codons are marked by solid arrowheads. The tRNA-like structure and tmRNA-encoded tag peptide are shown at the top and the bottom of the tmRNA model, respectively. The cross-linked sites of covalent attachment of tmRNA to ribosomal protein S1 are indicated by “lollipops” denoted xl-1 to xl-6.

10.1128/9781555818142/fig32-1_thmb.gif

10.1128/9781555818142/fig32-1.gif

Figure 1

E. coli tmRNA secondary structure with base pairs supported by comparative sequence analysis (Fox and Woese, 1975; Larsen and Zwieb, 1991). Perfect base pairs are connected with lines; G•U pairs are connected with open circles. Helices are highlighted in gray and numbered from 1 to 12 from the 5' end. Helical sections are given extensions with lowercase letters. The nucleosides are labeled with dots in increments of 10 and are numbered, if allowed by the available space. The 5'-to-3' direction of the single RNA chain is indicated by lines with open arrowheads. The four pseudoknots are marked pk1 to pk4. The solid star marks the beginning of the tmRNA coding region. In-frame stop codons are marked by solid arrowheads. The tRNA-like structure and tmRNA-encoded tag peptide are shown at the top and the bottom of the tmRNA model, respectively. The cross-linked sites of covalent attachment of tmRNA to ribosomal protein S1 are indicated by “lollipops” denoted xl-1 to xl-6.

Alignment of tmRNA-encoded tag sequences as deduced from the tmRNA alignment (Zwieb et al., 1999a). The sequences are ordered phylogenetically with abbreviated species names, as in the tmRDB (http://psyche.uthct.edu/dbs/tmRDB). Nonpolar amino acids are shown in reverse print; stop codons are indicated by stars.

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Figure 2

Alignment of tmRNA-encoded tag sequences as deduced from the tmRNA alignment (Zwieb et al., 1999a). The sequences are ordered phylogenetically with abbreviated species names, as in the tmRDB (http://psyche.uthct.edu/dbs/tmRDB). Nonpolar amino acids are shown in reverse print; stop codons are indicated by stars.

Mechanism of tmRNA in trans-translation. (a) The ribosome is stalled at the 3' end of mRNA lacking a stop codon. The incomplete polypeptide chain remains bound to the P-site tRNA. (b) Alanyl-tmRNA binds the ribosomal A site assisted by ribosomal protein S1 and (c) accepts the polypeptide from the peptidyl-tRNA. (d) After the release of deacylated tRNA and damaged mRNA, peptidyl-tmRNA is translocated to the P site. (e) Aminoacyl-tRNA binds the resume codon in the A site. (f) After transpeptidylation, peptidyl-tRNA translocates to the P site and tmRNA adopts the mRNA-mode. (g) Releasing factors (RF) recognize the stop codon at the A site, and (h) the trans-translational complex dissociates. The tagged protein is degraded by proteases in the cytoplasm (ClpAP and ClpXP), cell membrane (HflP), or periplasm (Tsp).

10.1128/9781555818142/fig32-3_thmb.gif

10.1128/9781555818142/fig32-3.gif

Figure 3

Mechanism of tmRNA in trans-translation. (a) The ribosome is stalled at the 3' end of mRNA lacking a stop codon. The incomplete polypeptide chain remains bound to the P-site tRNA. (b) Alanyl-tmRNA binds the ribosomal A site assisted by ribosomal protein S1 and (c) accepts the polypeptide from the peptidyl-tRNA. (d) After the release of deacylated tRNA and damaged mRNA, peptidyl-tmRNA is translocated to the P site. (e) Aminoacyl-tRNA binds the resume codon in the A site. (f) After transpeptidylation, peptidyl-tRNA translocates to the P site and tmRNA adopts the mRNA-mode. (g) Releasing factors (RF) recognize the stop codon at the A site, and (h) the trans-translational complex dissociates. The tagged protein is degraded by proteases in the cytoplasm (ClpAP and ClpXP), cell membrane (HflP), or periplasm (Tsp).